Two-site immunochemiluminometric assay of intact human parathyrin in serum with use of a tracer peptide purified by reversed-phase high-performance liquid chromatography

Abstract

For this two-site immunochemiluminometric assay of intact human parathyrin (hPTH), the luminescent tracer was synthetic hPTH(53-84), conjugated via succinimide linkage to (aminobutyl)ethyl-isoluminol hemisuccinimide (ABEI-H). Purification of the labeled hPTH(53-84) by reversed-phase high-performance liquid chromatography allowed isolation of the conjugate having the highest incorporation of ABEI-H, 1.6 mol per mole of hPTH(53-84). The solid-phase antibody directed against the N-terminal part of hPTH was immobilized by adsorption onto the polystyrene surface of the assay tube and extracted the intact hPTH and N-terminal fragments. Another antibody against synthetic hPTH(53-84), which bound to the C-terminal part of intact hPTH, was indirectly labeled as its second free binding site with the ABEI-H-labeled hPTH(53-84). The assay has a detection limit of 0.5 pmol/L; it is accurate, precise, and reliable; and it shows a linear response for samples containing up to 100 pmol of hPTH per liter. The normal reference interval ranged from 1.8 to 5.9 pmol/L; 56 patients with primary hyperparathyroidism had concentrations ranging from 5.9 to 113 pmol/L. The concentrations detected in patients with idiopathic hypoparathyroidism were below the normal reference interval.